Quantification of maceration changes using post mortem MRI in fetuses.

BACKGROUND: Post mortem imaging is playing an increasingly important role in perinatal autopsy, and correct interpretation of imaging changes is paramount. This is particularly important following intra-uterine fetal death, where there may be fetal maceration. The aim of this study was to investigate whether any changes seen on a whole body fetal post mortem magnetic resonance imaging (PMMR) correspond to maceration at conventional autopsy. METHODS: We performed pre-autopsy PMMR in 75 fetuses using a 1.5 Tesla Siemens Avanto MR scanner (Erlangen, Germany). PMMR images were reported blinded to the clinical history and autopsy data using a numerical severity scale (0 = no maceration changes to 2 = severe maceration changes) for 6 different visceral organs (total 12). The degree of maceration at autopsy was categorized according to severity on a numerical scale (1 = no maceration to 4 = severe maceration). We also generated quantitative maps to measure the liver and lung T2. RESULTS: The mean PMMR maceration score correlated well with the autopsy maceration score (R(2) = 0.93). A PMMR score of ≥4.5 had a sensitivity of 91%, specificity of 64%, for detecting moderate or severe maceration at autopsy. Liver and lung T2 were increased in fetuses with maceration scores of 3-4 in comparison to those with 1-2 (liver p = 0.03, lung p = 0.02). CONCLUSIONS: There was a good correlation between PMMR maceration score and the extent of maceration seen at conventional autopsy. This score may be useful in interpretation of fetal PMMR.

Effects of saliva substitutes on oral status in patients with Type 2 diabetes.

AIMS: To assess oral status in a sample of Type 2 diabetic patients before and after therapy with saliva substitutes and oral status in a control group of diabetic patients who were not given saliva substitutes. METHODS: Salivary flow rate was determined in 134 patients (mean age 47.9 ± 2.9 years) with Type 2 diabetes. Mean salivary rate was significantly low compared with a healthy control group. The sample of 134 patients was randomly divided into two groups of 67 people each. One group was given immunologically active salivary substitutes for 6 months, the other group was given nothing. Each patient of the two groups underwent a dental and periodontal examination at the beginning of the study and 6 months later. RESULTS: As regards carious teeth and teeth loss, there was no statistical difference between the first group after 6 months of treatment with salivary substitutes and the control group (P>0.01). Salivary substitutes did not significantly reduce the periodontal disease (P>0.01). In the group treated with salivary substitutes, after 6 months of therapy, the average dental plaque index decreased from 2.3 ± 0.73 to 1.6 ± 0.56, patients with gingivitis decreased from 66 to 43% and patients with positive yeast counts decreased from 60 to 37%. These differences were statistically significant (P<0.01). CONCLUSIONS: In Type 2 diabetes, in the case of hyposalivation, a therapy with immunologically active saliva substitutes can be of help in reducing the amount of plaque, gingivitis and positive yeast counts.

Prospective research on infants with mild encephalopathy: the PRIME study.

OBJECTIVE: To determine short-term outcomes of infants with evidence of hypoxia-ischemia at birth and classified as mild neonatal encephalopathy (NE) at <6 h of age. STUDY DESIGN: Prospective multicenter study. Mild NE was defined as ⩾1 abnormal category in modified Sarnat score. Primary outcome was any abnormality on early amplitude integrated electroencephalogram (aEEG) or seizures, abnormal brain magnetic resonance imaging (MRI) or neurological exam at discharge. RESULTS: A total of 54/63 (86%) of enrolled infants had data on components of the primary outcome, which was abnormal in 28/54 (52%): discontinuous aEEG (n=4), MRI (n=9) and discharge exam (n=22). Abnormal tone and/or incomplete Moro were the most common findings. MRI abnormalities were confined to cerebral cortex but two infants had basal ganglia and/or thalamus involvement. The 18 to 24 months follow-up is ongoing. CONCLUSIONS: A larger than expected proportion of mild NE infants with abnormal outcomes was observed. Future research should evaluate safety and efficacy of neuroprotection for mild NE.

Enhancement of oleyl alcohol anti tumor activity through complexation in polyvinylalcohol amphiphilic derivatives.

Oleyl alcohol was complexed with new amphiphilic polyvinylalcohol derivatives with the aim of increasing its aqueous solubility, thus improving bioavailability and favoring its antitumor activity. Water-soluble amphiphilic polymers were prepared by polyvinyl alcohol (PVA) substitution with oleyl chains through a succinyl spacer at 2% and 3% substitution degree. The complexes were obtained by spray-drying hydroalcoholic solutions of the substituted polymers and free oleyl alcohol at different weight ratios (3:1; 5:1; 10:1 w/w). The main physicochemical characteristics of the complexes were analyzed and correlated to the cytotoxic activity of oleyl alcohol toward tumor cell lines. The complexes strongly increased the aqueous solubility of oleyl alcohol and provided oleyl alcohol release in the presence of extractive conditions (simulating in vivo absorption). The complexes obtained by 10:1 polymer:fatty alcohol weight ratio offered higher release rates than the 5:1 and 3:1 ratios, respectively. Complexation also increased oleyl alcohol cytotoxicity toward tumor cells due to increased availability of the active molecule in the aqueous phase. Pure polymers were found to be biocompatible and no toxic effect was detected up to the highest concentration used in the present study (500 mu g/ml). The complexation of oleyl alcohol with the polymers analyzed here efficiently increased the availability of the fatty alcohol in aqueous environment. The enhanced cytotoxicity toward tumor cells of the complexed oleyl alcohol and the polymer biocompatibility make these amphiphilic PVA derivatives interesting candidates for soluble pharmaceutical formulations containing hydrophobic drugs whose therapeutic potential is often underestimated due to unsuitable levels of their aqueous solubilization.

Predictive value of amplitude-integrated EEG (aEEG) after rescue hypothermic neuroprotection for hypoxic ischemic encephalopathy: a meta-analysis.

OBJECTIVE: Amplitude-integrated electroencephalography (aEEG) is a useful bedside tool in predicting the neurodevelopmental outcome after neonatal encephalopathy; however, the prognostic accuracy may be altered by rescue hypothermic neuroprotection. The objective of this study is to examine the prognostic accuracy of aEEG for predicting long-term neurodevelopmental outcomes in term newborn infants undergoing therapeutic hypothermia for neonatal encephalopathy. STUDY DESIGN: We examined all studies (Medline, Cumulative Index to Nursing and Allied Health Literature and the Cochrane Library; 2000 to 2014) comparing aEEG (6, 24, 48 or 72 h) in term encephalopathic babies undergoing therapeutic hypothermia, with neurodevelopmental outcome at 1 year or more. We extracted individual patient data from the eligible studies to calculate prognostic indices with exact confidence intervals (CIs). We considered continuous normal voltage as normal aEEG pattern and discontinuous normal voltage, burst suppression, flat trace and persistently low voltage as abnormal, and defined adverse outcome as death or moderate/severe disability at 1 year. RESULTS: We reviewed a total of 70 articles, 17 of which met the inclusion criteria. Eight studies were excluded and 9 studies (N=520) were included in the meta-analysis. The pooled sensitivity and specificity for an abnormal trace at 6 h of age to predict adverse outcome were 96% (95% CI 91 to 98%) and 39% (95% CI 32 to 46%). The diagnostic odds ratio of an abnormal trace was highest at 48 h (66.9 (95% CI 19.7, 227.2)). CONCLUSIONS: A persistantly abnormal aEEG at 48 h or more is associated with an adverse neurodevelopmal outcome. The positive prognostic value of 6 h aEEG is poor and good outcome may occur despite abnormal aEEG. Conversely, a normal 6 h aEEG has a good negative predictive value although do not exclude adverse outcomes.

Synergistic differentiation-promoting activity of interferon gamma and tumor necrosis factor-alpha: role of receptor regulation on human neuroblasts.

BACKGROUND: Interferon gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF) synergize in inducing human neuroblastoma cells to differentiate terminally in vitro into mature nonproliferating neurons. The mechanisms by which this synergistic activity takes place are still obscure. PURPOSE: To understand the basis of IFN-gamma-TNF synergism, we investigated the constitutive equipment of receptors to IFN-gamma and TNF in two human neuroblastoma cell lines (i.e., LAN-5 and GI-LI-N) and their quantitative and functional variations following treatment with IFN-gamma or TNF. METHODS: IFN-gamma receptors and TNF receptors were assessed and functionally characterized by radioreceptor-binding assay before and after treatment of the cells with IFN-gamma or TNF. The TNF receptor subtypes were identified by the reverse transcriptase-polymerase chain reaction, chemical cross-linking of receptors to iodinated TNF, and inhibition of TNF binding by type-specific anti-TNF receptor monoclonal antibodies. The effects of cytokines on cell differentiation were assessed by thymidine incorporation inhibition and morphologic maturation. RESULTS: No quantitative or functional modification of IFN-gamma receptors was observed in TNF-treated cells. However, after treatment with IFN-gamma, TNF receptor numbers were enhanced to a different extent in both cell lines. The two neuroblastoma cell lines expressed, both constitutively and after IFN-gamma induction, only one species of TNF receptor, i.e., the p80 form in LAN-5 and the p60 form in GI-LI-N. Sequential treatment with IFN-gamma followed by TNF, but not in the opposite order, could reproduce the early effects of differentiation in neuroblastoma cells, supporting a role for TNF receptor up-regulation as a basis for the cooperation between the two cytokines. CONCLUSION: The results strongly suggest that receptor regulation can be at least one mechanism by which IFN-gamma and TNF exert their synergistic effects. Moreover, it appears that the two TNF receptor types are redundant in signaling neuroblastoma cell differentiation. IMPLICATIONS: Our findings can provide a guideline for a rational design of experimental differentiation-based therapeutic protocols in patients with neuroblastoma.

Delivery of c-myb antisense oligodeoxynucleotides to human neuroblastoma cells via disialoganglioside GD(2)-targeted immunoliposomes: antitumor effects.

BACKGROUND: Advanced-stage neuroblastoma resists conventional treatment; hence, novel therapeutic approaches are required. We evaluated the use of c-myb antisense oligodeoxynucleotides (asODNs) delivered to cells via targeted immunoliposomes to inhibit c-Myb protein expression and neuroblastoma cell proliferation in vitro. METHODS: Phosphorothioate asODNs and control sequences were encapsulated in cationic lipid, and the resulting particles were coated with neutral lipids to produce coated cationic liposomes (CCLs). Monoclonal antibodies directed against the disialoganglioside GD(2) were covalently coupled to the CCLs. (3)H-labeled liposomes were used to measure cellular binding, and cellular uptake of asODNs was evaluated by dot-blot analysis. Growth inhibition was quantified by counting trypan blue dye-stained cells. Expression of c-Myb protein was examined by western blot analysis. RESULTS: Our methods produced GD(2)-targeted liposomes that stably entrapped 80%-90% of added c-myb asODNs. These liposomes showed concentration-dependent binding to GD(2)-positive neuroblastoma cells that could be blocked by soluble anti-GD(2) monoclonal antibodies. GD(2)-targeted liposomes increased the uptake of asODNs by neuroblastoma cells by a factor of fourfold to 10-fold over that obtained with free asODNs. Neuroblastoma cell proliferation was inhibited to a greater extent by GD(2)-targeted liposomes containing c-myb asODNs than by nontargeted liposomes or free asODNs. GD(2)-targeted liposomes containing c-myb asODNs specifically reduced expression of c-Myb protein by neuroblastoma cells. Enhanced liposome binding and asODN uptake, as well as the antiproliferative effect, were not evident in GD(2)-negative cells. CONCLUSIONS: Encapsulation of asODNs into immunoliposomes appears to enhance their toxicity toward targeted cells while shielding nontargeted cells from antisense effects and may be efficacious for the delivery of drugs with broad therapeutic applications to tumor cells.

Accumulation of m-iodobenzylguanidine by neuroblastoma cells results from independent uptake and storage mechanisms.

The modalities of uptake and storage of iodine-labeled m-iodobenzylguanidine (MIBG) by four human neuroblastoma cell lines have been studied. SK-N-BE(2)C cell line has been shown to possess the specific (type 1) MIBG uptake, as well as an efficient extravesicular storage mechanism. Conversely, LAN-5 cells, which show a nonsaturation kinetic of MIBG incorporation, lack only the ability to efficiently store the MIBG taken up by a mechanism that can be pharmacologically defined as uptake 1. The two other neuroblastoma cell lines tested, GI-LI-N and GI-CA-N, lack both uptake and storage capacity. In view of the fact that the only detailed study on specific MIBG uptake by a human neuroblastoma cell line has been carried out on SK-N-SH, a highly heterogeneous cell line, our report provides new insights on the molecular and cellular pharmacology of radiolabeled MIBG.

The combination of gamma-interferon and tumor necrosis factor causes a rapid and extensive differentiation of human neuroblastoma cells.

Neuroblastoma (NB), a tumor originating from the sympathetic nervous system, is the most common extracranial neurological tumor of childhood. Human NB cells may differentiate in vitro under treatment with biological agents, as gamma-interferon (IFN-gamma) and tumor necrosis factor (TNF). Unfortunately, NB cell lines resistant to the differentiation-inducing effects of both drugs have been observed. Here we demonstrate that a combination of IFN-gamma plus TNF causes extensive and generalized differentiation of NB cells toward a neuronal phenotype. Both IFN-gamma and TNF, given alone, moderately reduced cell growth and induced partial morphological maturation. Their combination further reduced cell proliferation. The combined treatment gave a synergistic rather than additive cytostatic effect, documented also by a dramatically enhanced differentiation toward a neuronal morphology. Membrane immunofluorescence showed a homologous and heterologous up-regulation of IFN-gamma receptor, as well as a marked induction of HLA Class I antigens and, to a lesser extent, of Class II antigens on NB cells induced to differentiate. Treatment of NB cell lines with IFN-gamma/TNF results in the induction of a differentiated phenotype, as indicated by the increased expression of the Mr 160,000 and 200,000 neurofilament proteins and that of microtubule-associated proteins. Evaluation of biochemical markers of neuronal differentiation confirmed the ability of the combined treatment to induce neuroblast maturation. These results suggest that the combination of IFN-gamma and TNF should be considered for experimental clinical trials in neuroblastoma.

gamma-Interferon increases metaiodobenzylguanidine incorporation and retention in human neuroblastoma cells.

Iodine-labeled m-iodobenzylguanidine (MIBG) is a widely used radiopharmaceutical for both diagnosis and biologically targeted radiotherapy of neuroblastoma. However, resistance to the radiotherapeutic effects of MIBG is often encountered, mainly due to lack of MIBG accumulation by neoplastic cells. We have investigated whether the induction of neuroblastoma cell differentiation modifies MIBG incorporation and retention. LAN-5 cells were selected, due to their moderate ability to take up MIBG. Treatment of these cells with gamma-interferon (IFN-gamma) resulted in morphological changes accompanied by a significant increase in overall cell-associated MIBG. Desimipramine, but not reserpine, easily depleted IFN-gamma-treated LAN-5 cells of their MIBG content. This suggests that the mechanism involved is an uptake enhancement rather than an improved storage ability. Indeed, IFN-gamma induces de nov synthesis of MIBG receptor-transporters, as demonstrated by polymerase chain reaction amplification and semiquantitative analysis. Our results suggest that pretreating neuroblastoma patients with IFN-gamma before MIBG administration may enhance the efficacy of both biologically targeted radioimaging and therapy of this tumor.

Differential effects of N-(4-hydroxyphenyl)retinamide and retinoic acid on neuroblastoma cells: apoptosis versus differentiation.

Retinoids exert various important biological effects in the control of normal growth, differentiation, and fetal development. While retinoic acid (RA) has entered clinical trials as a differentiation-promoting agent, it is only recently that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has been shown to be of potential clinical interest in cancer chemoprevention and treatment. Since thus far no data exist on the effects of HPR on neural crest cell-derived tumors, we have examined its in vitro effects on neuroblastoma (NB) cell lines and found that at relevant pharmacological concentrations it induces a dose-dependent growth inhibition. The antiproliferative effects of HPR were, in six of six cell lines tested, drastically more potent that those induced by an equimolar dose of RA. Time course growth analysis showed that HPR at 3 x 10(-6) M induces a very rapid (24-72 h) fall in thymidine uptake (> 90%), whereas at 3 x 10(-7) M it exhibits cytostatic effects. In contrast to RA, HPR did not show morphological changes typical of NB cell maturation nor did it induce the expression of any cytoskeletal protein associated with neuronal differentiation. DNA flow cytofluorimetric analysis revealed that HPR did not induce an arrest in a specific phase of the cell cycle while triggering apoptosis. This phenomenon was evidenced both by the visualization of "DNA ladders" on gel electrophoresis and by a quantitative assay for evaluating programmed cell death based upon the labeling of DNA breaks with tritiated thymidine. With the latter method, apoptotic cells were detectable as early as 3-6 h after treatment of NB cells with 10(-5) M HPR, while more than 50% of cells were apoptotic by 24-72 h following exposure to 3 x 10(-6) M HPR. In contrast, RA induced a low rate of apoptosis in NB cells only after 3-5 days. Time lapse photomicroscopy showed that NB cells treated with HPR underwent a death process highly reminiscent of apoptosis, with progressive condensation of the cytoplasm around the nucleus and intense cell shrinkage. The cells then rounded up and detached from the plate. Furthermore, propidium iodide staining of the DNA showed that a high proportion of cells treated with HPR displayed a small and brightly staining nucleus; chromatin appeared aggregated into dense masses in the nuclear periphery, a typical feature of apoptotic cells. In conclusion, our study demonstrates that contrary to the differentiation-promoting activity of RA, HPR dramatically suppresses NB cell growth by inducing programmed cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of differentiation and apoptosis by interferon-gamma in human neuroblastoma cells in vitro as a dual and alternative early biological response.

Interferon-gamma (IFN-gamma) has a well known differentiation-promoting activity on several neuroblastoma (NB) cell lines and has also been reported to induce apoptosis in different cellular models. We have investigated the potential of IFN-gamma to trigger, besides differentiation, programmed cell death in NB cells and the relationship between these processes. Nine NB cell lines, characterized by different phenotypic and maturational features, were cultured in the presence of IFN-gamma (1000 IU/ml) for up to 5 days with either only one treatment at the start of the culture or renewing the culture medium (with or without IFN-gamma) every other day. Neuronal differentiation was assessed by evaluation of morphological changes and expression of mature cytoskeletal proteins, while apoptosis was evaluated at the desired times by fluorescent and electronic microscopy, DNA content analysis and DNA fragmentation assay. Our findings show that apoptosis is an early (mainly non post-differentiative) event and is much more evident following a single IFN-gamma administration. Moreover, IFN-gamma-triggered apoptosis is independent of the cellular phenotype (schwannian or neuronal) and appears to be mutually exclusive with respect to differentiation at the single cell level. Our results strengthen the potential of IFN-gamma as a promising therapeutic agent for NB.

Modified polyvinylalcohol for encapsulation of all-trans-retinoic acid in polymeric micelles.

All-trans-retinoic acid (ATRA) is now included in many antitumor therapeutic schemes for the treatment of acute promyelocytic leukaemia, Kaposi's sarcoma, head and neck squamous cell carcinoma, ovarian carcinoma, bladder cancer and neuroblastoma. Unfortunately its poor aqueous solubility hampers its parenteral formulation. To date, there is no parenteral formulation of ATRA commercially available and oral administration of ATRA is associated with progressively diminishing ATRA levels in plasma, which is related to induction of retinoic acid-binding protein and increased drug catabolism by cytochrome P-450-mediated reaction. An ATRA formulation, obtained by complexation of the drug into polymeric micelles, might be suitable for parenteral administration overcoming these unwanted effects. To this purpose we prepared an amphiphilic polymer by polyvinylalcohol (PVA) substitution with oleyl amine at 1.5% substitution degree (mol substituent per 100 mol hydroxyvinylmonomer) and evaluated its functional properties with regard to ATRA complexation. The substituted polymer displayed ability to interact with ATRA both in aqueous solution and in the solid state following spray-drying of drug-polymer hydro-alcoholic solutions. The spray-dried complexes rapidly dissolved in water providing high levels of ATRA solubilization as a function of the drug-polymer weight ratio. The complexes characterized by 1:5 drug-polymer weight ratio provided higher levels of ATRA solubilization than 1:3 and 1:10 drug-polymer weight ratios respectively. Pre-formed polymeric micelles in water equilibrated in the presence of excess solid ATRA provided the lowest levels of solubilization. The drug release from the complexes was very slow in PBS, indicating their suitability in antitumor drug targeting where a fundamental requirement is stability towards drug release for at least 24 h, corresponding to the average circulation time period of macromolecular carriers. The cytotoxicity studies against neuroblastoma cell lines outlined increased cytotoxicity of complexed ATRA with respect to free ATRA, likely due to the increased bioavailability of the hydrophobic drug from the complex. We conclude that ATRA entrapped into self-assembling polymer micelles may be a useful parenteral ATRA formulation overcoming the unwanted pharmacological mechanism that lead to acquired retinoid resistance.

Modified doxorubicin for improved encapsulation in PVA polymeric micelles.

Polyvinylalcohol, partially substituted with lipophilic acyl chains, generates polymeric micelles in aqueous phase, containing a hydrophobic core able to encapsulate lipophilic drugs. Two types of polymers were obtained by conjugation of polyvinylalcohol with oleoyl or linoleoyl chains as pendant groups. The polymers, at a substitution degree of approximately 1%, are soluble in water and form polymeric micelles whose size increases with polymer concentration. Doxorubicin was hydrophobized, by linking an oleoyl chain via amide bond, to make the drug more similar to the substituted polymers and promote its encapsulation into the inner core of the micelles. The properties of the drug-polymer systems were evaluated in solution by dynamic light scattering technique and correlated to the physicochemical characteristics of the drug and the substituted polymers. Solubilization tests revealed that the similarity of the chain, in both the polymer and the drug, promotes better drug encapsulation in the oleoyl than linoleoyl derivative. The drug-polymer systems are stable in phosphate buffer saline (pH 7.4) at 37 degrees C, and the release of the drug is activated by the presence of the proteolytic enzyme pronase-E. The enzyme activated drug release and the size of the polymeric micelles, compatible with the pore dimensions of the tumor vessels, make these systems interesting for targeting lipophilic drugs to solid tumors, where the proteolytic enzyme concentration strongly raises with respect to the other body compartments.

Stimulation of receptor-coupled phospholipase A2 by interferon-gamma.

The biomolecular mechanisms that mediate signal transduction by type II (gamma) interferon (IFN) are poorly understood. IFN-gamma is a potent growth inhibitory cytokine also endowed with antiviral, immunomodulatory, and differentiating activities on various cell targets, including neural cells. IFN-gamma induced a rapid and transient activation of phospholipase A2 in LAN-5, a human neuroblastoma cell line. A consequence of phospholipase A2 activation was the release of arachidonic acid and the generation of lysophospholipids from membrane phospholipids. Treatment of pre-labeled LAN-5 cells with a receptor-saturating concentration of IFN-gamma led to a time-dependent release of [3H]arachidonic acid into the culture media and generation of [32P]lysophosphatidylcholine. Pretreatment of cultures with the phospholipase A2 inhibitor, bromophenacyl bromide, markedly inhibited both [3H]arachidonic acid release and lysophosphatidylcholine production induced by IFN-gamma treatment. Pretreatment of LAN-5 cells with nordihydroguaiaretic acid, a lipoxygenase inhibitor, or with indomethacin, a cyclooxygenase inhibitor, amplified the release of [3H]arachidonic acid and production of lysophosphatidylcholine induced by non-saturating concentrations of IFN-gamma. In parallel, and with the same time-dependent effect, a significant decrease in phosphatidylcholine labeling was observed in IFN-gamma-treated cells, further indicating that a potential signal transduction mechanism of IFN-gamma is the hydrolysis of membrane phosphatidylcholine by phospholipase A2.

Angiogenesis and anti-angiogenesis in neuroblastoma.

Angiogenesis is a biological process by which new capillaries are formed from pre-existing vessels. It occurs in physiological and pathological conditions, such as tumours, where a specific critical turning point is the transition from the avascular to the vascular phase. Tumour angiogenesis depends mainly on the release by neoplastic cells of growth factors specific for endothelial cells that able to stimulate the growth of the host's blood vessels. This review summarises the literature concerning the relationship between angiogenesis and progression in human neuroblastoma, the most common extracranial solid tumour of infancy and childhood. It is becoming increasingly evident that agents which interfere with blood vessel formation also block tumour progression. Accordingly, anti-angiogenic tumour therapy has gained much interest in preclinical and clinical assessments. The recent applications of anti-angiogenic agents which interfere or block neuroblastoma progression are reviewed.

Circadian variations of autologous mixed lymphocyte reactions and endogenous cortisol.

The degree of proliferation of human T cells stimulated with autologous PHA-T cells and with autologous non-T cells displays circadian variations. The highest proliferation occurs with cells isolated from blood drawn at 8 a.m. in mixed lymphocyte reactions (MLR) with autologous PHA-T cells and from blood drawn at 8 p.m. in MLR with autologous non-T cells. The circadian variations of autologous MLRs appear to reflect changes in the proliferative response of T cells. In autologous MLRs with non-T cells as stimulators the extent of proliferation was inversely correlated with the level of endogenous cortisol. The circadian variations of autologous MLRs do not reflect non-specific changes in the proliferative and stimulatory properties of T and non-T cells, since circadian variations were not observed in the proliferative response of T cells to mitogens and in allogeneic MLRs. Circadian variations of autologous MLRs must be taken into account when analyzing abnormalities of these reactions in pathological conditions.

Immunoliposomal fenretinide: a novel antitumoral drug for human neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In advanced disease stages, prognosis is poor and treatments have limited efficacy, thus novel strategies are warranted. The synthetic retinoid fenretinide (HPR) induces apoptosis in NB and melanoma cell lines. We reported an in vitro potentiation of HPR effects on melanoma cells when the drug is incorporated into GD2-targeted immunoliposomes (anti-GD2-SIL-HPR). Here, we investigated the antitumor activity of anti-GD2-SIL-HPR against NB cells, both in vitro and in vivo. Anti-GD2-immunoliposomes (anti-GD2-SIL) showed specific, competitive binding to, and uptake by, various NB cell lines. Moreover, anti-GD2-SIL-HPR presented increased selectivity and efficacy in inhibiting NB cell proliferation through the induction of apoptosis, compared to free drug and SL-HPR. In an in vivo NB metastatic model, we demonstrated that anti-GD2-SIL-HPR completely inhibited the development of macroscopic and microscopic metastases in comparison to controls. However, similar, but significantly less potent antitumor effect was observed also in mice treated with anti-GD2 immunoliposomes without HPR (anti-GD2-SIL-blank) or anti-GD2 mAb alone (P=0.0297 and P=0.0294, respectively, vs. anti-GD2-SIL-HPR). Moreover, our results clearly demonstrated that, although anti-GD2 mAb had a strong antitumor effect in this in vivo NB model, 100% curability was obtained only following treatment with anti-GD2-SIL-HPR (P<0.0001). Anti-GD2 liposomal HPR should receive clinical evaluation as adjuvant therapy of neuroblastoma.

Fenretinide as an anti-angiogenic agent in neuroblastoma.

Angiogenesis is a critical event in the progression of human neuroblastoma. This mini-review summarizes our literature and experimental data concerning the use of anti-angiogenic molecules, such as TNP-470 and fenretinide, in neuroblastoma treatment.

Anti-GD2 monoclonal antibody immunotherapy: a promising strategy in the prevention of neuroblastoma relapse.

In spite of the satisfactory frequency of clinical response to first-line therapy in neuroblastoma (NB), complete eradication of NB cells is rarely achieved. As a consequence, the majority of patients with advanced stage NB undergo relapse, which is often resistant to conventional treatment and rapidly overwhelming. Thus, after induction of the apparent remission, new therapeutic strategies are needed to completely eradicate the small number of surviving NB cells and to prevent relapse. We explored the potential of different doses of the anti-GD2 monoclonal antibody (mAb) 14G2a in an experimental metastatic model where a limited number of HTLA-230 human NB cells are injected i.v. into nude mice, leading to extensive metastases and death of animals within 7-8 weeks. Treatment with 14G2a mAb (1-4 mg/kg cumulative dose given as five i.v. daily administrations) dramatically reduced the metastatic spread of NB cells and prolonged the long-term survival of treated mice in a dose-dependent manner. Neither macrophages nor NK cells appeared to contribute to the protective effect of antibody treatment in vivo, suggesting either an involvement of granulocytes or a complement-mediated cytotoxicity towards NB cells. Whatever the effecting mechanism(s) involved, these results strongly support the clinical use of anti-GD2 mAbs after first-line induction regimens.